Glucocorticoids (or simply steroids) remain the primary therapy for treating asthma. The glucocorticoid receptor (GR) is a potent transcription factor that, in response to glucocorticoids, can bind to DNA and directly induce the expression of numerous downstream genes. However, a distinct mechanism in which GR tethers to and represses inflammatory transcription factors such as NFkB (i.e. ?transrepression?) has long been viewed as central to steroid efficacy in asthma. Despite this dogma, airway cell data directly supporting the transrepression model are limited, and there remains considerable uncertainty regarding the mechanistic underpinning of steroid activity in the asthmatic airway. This knowledge gap has hampered efforts to improve steroid-based therapies, which cause debilitating side effects when used systemically to treat severe asthma. In this proposal, we will test the innovative hypothesis that therapeutic effects of steroids in airway smooth muscle (ASM), which exhibits extensive remodeling and cytokine secretion in difficult-to-treat asthma, are mediated through GR-based gene induction. Supporting this notion, our preliminary data has defined a pathway in which GR induces the Kruppel-like transcription factor, KLF15, which we have shown is a potent repressor of ASM remodeling. Our data further suggest that GR and KLF15 cooperatively induce CDKN1C, which is a cell cycle inhibitor, and PLCD1, a cytoskeletal regulator that appears to block smooth muscle actin expression in ASM. Thus, our data implicate steroid-induced pathways and cooperation between GR and KLF15 as potentially repressing pathologic ASM remodeling. Moreover, our data indicate that steroid-mediated repression of ASM cytokine expression also involves GR-mediated gene induction, in this case through a surprising cooperative relationship between GR and the prototypical pro-inflammatory transcription factor, NFB. Indeed, entirely orthogonal to the transrepression model, our data suggest that GR cooperates with NF?B to induce the expression of key anti-inflammatory genes such as A20, which is a potent negative feedback regulator of TNF and TLR signaling whose dysfunction is implicated in promoting asthma pathogenesis. Thus, cooperation between GR and factors such as KLF15 and NFkB, leading to gene induction, appears to be a central mechanism of steroid activity in ASM. However, the mechanistic basis for induction of CDKN1C and PLCD1 by GR and KLF15 has yet to be defined, and the precise role of these targets in repressing or reversing asthmatic ASM remodeling remains to be elucidated, Similarly, although our data support a role for GR-NFkB cooperative regulation of A20 in repressing ASM cytokine secretion, the genome-wide extent of GR- NFkB cooperation in ASM, and the in vivo consequences of GR-NFkB cooperation are not known. Thus, the goals of this proposal are: (1) to determine the mechanistic basis and consequences of GR-KLF15 cooperative gene induction, with a specific focus on regulation and phenotypic effects of CDKN1C and PLCD1 in ASM remodeling; (2) to determine the role of cooperation between GR and NFkB in mediating cytokine repression in ASM, with a specific focus on GR-NFkB cooperative regulation of A20 and the ASM-specific role of A20 in vivo in murine models of allergic airway disease; and (3) determine whether ASM from asthma patients exhibits perturbations in these novel pathways controlled by GR-mediated gene induction. In addition, through studying freshly isolated ASM from donor lungs, we will provide important insights into correlations between fresh and cultured ASM, particularly with respect to steroid-mediated gene induction and resistance. Taken together, this proposal will provide detailed molecular insight into the mechanisms underpinning GR function in ASM. This has broad implications for developing novel asthma therapeutics that selectively target severe airway remodeling and refractory inflammation in severe asthma.